Proteins are at the crossroads of all biological processes including molecular recognition, catalysis, signal transduction, information storage and mobility. Yet, we have little understanding of the factors that control the structure, stability, folding and function of proteins. X-ray crystallography and site-directed mutagenesis have proven to be extremely powerful tools for analyzing protein structure/function. However the restrictions imposed on the latter technique by the natural twenty amino acids limit its usefulness. Recently, we have succeeded in developing a general biosynthetic method which, for the first time, makes it possible to site-specifically incorporate unnatural amino acids with novel steric and electronic properties into proteins. Our ability to insert amino acids with a wide range of side-chain and backbone structures into proteins allows us for the first time to carry out precise "physical organic" chemistry on this important class of molecules. Our objectives during the next three years will focus (a) on testing the scope of this new methodology with regard to the kind of amino acid structures that can be incorporated into proteins and (b) using this methodology to analyze protein structure and function. We will (i) undertake a systematic study of those factors that determine protein stability in the model system, T4 lysozyme, including the effects of hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals interactions and entropy on protein thermal stability and (ii) explore the catalytic mechanism of staphylococcal nuclease in an effort to understand in greater detail how this enzyme achieves its remarkable catalytic advantage. In addition, we will develop new tools for analyzing protein structures (folded and unfolded), protein-protein interactions and conformational changes including the site specific incorporation of fluorescent, spin labelled and isotopically labelled amino acids for biophysical studies of protein structure.